Bottom Line:
Efforts focused solely on the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein may not optimize neutralizing antibody (NAb) responses.Here we show that immunogens based on full-length S DNA and S1 subunit protein elicit robust serum-neutralizing activity against several MERS-CoV strains in mice and non-human primates.Multiple neutralization mechanisms were demonstrated by solving the atomic structure of a NAb-RBD complex, through sequencing of neutralization escape viruses and by constructing MERS-CoV S variants for serological assays.

f4: MERS-CoV-neutralizing mAbs target multiple regions of the RBD.Vaccine-induced mAb D12 binds to the DPP4-interacting region of the viral Spike RBD, blocking receptor binding. (a) (Left) Comparison of RBD binding to D12 and DPP4. RBD (cyan) with receptor-binding motif (residues 484–567, magenta) and D12 are shown in ribbon representation. The D12 heavy chain is light blue and the light chain is light green. The heavy chain complementarity determining regions (CDR) are light blue (CDR H1), blue (CDR H2) and dark blue (CDR H3), while the light chain CDRs are cyan (CDR L2), green (CDR L2) and pale yellow (CDR L3). The main interacting regions are in CDR H2, CDR H3 and CDR L2. (Right) DPP4 is shown in ribbon representation (green) with Asn 229 and the attached N-glycan (yellow) shown as sticks. The RBD is oriented as shown in the left panel. (b) Antibody:RBD and DPP4:RBD crystal structure complexes. RBD in surface representation is shown with the D12 heavy and light chain binding region coloured blue and green, respectively. The CDR loops are shown as ribbons and coloured as in (a; left). The rotated RBD shows the full D12 paratope: D12 CDR H2 interacts with RBD W535 and E536 residues, which predominantly interact with the Asn 229-associated N-glycan on DPP4 (centre). RBD, shown in surface representation with the DPP4-interacting region coloured green. Major interacting regions of DPP4 are shown as ribbon representations with Asn 229 and N-glycan shown as sticks (Supplementary Fig S11; right). (c) D12 and RBD interface. All CDRs are shown in ribbon representation, with interacting residues shown as sticks, and hydrogen bonds represented by dotted lines. (d,e) Crystal structure of MERS England1 RBD and effect of critical RBD mutations on binding. RBD residues 506 and 509, identified by mutagenesis analysis, are highlighted in green. Critical RBD residues 532, 535 and 536, identified by structural definition and viral resistance evolution, reduce or eliminate D12 binding (shown in red). ELISA results show that this set of mutations eliminate F11 or D12 binding.

Mentions:
Although the two most potent neutralizing mAbs—D12 and F11—targeted the RBD, their neutralization profiles were different, when mAb neutralization was tested against the panel of eight pseudotyped reporter viruses (Fig. 1c). Notably, F11 was unable to neutralize the Bisha1 strain (GenBank ID: KF600620.1) of MERS-CoV (Supplementary Fig. 9b), which differs from other strains by an aspartic acid to glycine substitution at residue 509, rendering it resistant to F11 while still susceptible to D12 neutralization. This finding was recapitulated in a pseudotyped virus neutralization assay where F11 neutralization activity against wild-type England1 was ablated with the introduction of a D509G mutation (Supplementary Fig. 9b). D12, in contrast, neutralized both viruses irrespective of the amino-acid change at position 509. In addition, the RBD 509G mutation abrogated F11 binding using enzyme-linked immunosorbent assay (ELISA) but did not affect D12 binding (Fig. 4e).

f4: MERS-CoV-neutralizing mAbs target multiple regions of the RBD.Vaccine-induced mAb D12 binds to the DPP4-interacting region of the viral Spike RBD, blocking receptor binding. (a) (Left) Comparison of RBD binding to D12 and DPP4. RBD (cyan) with receptor-binding motif (residues 484–567, magenta) and D12 are shown in ribbon representation. The D12 heavy chain is light blue and the light chain is light green. The heavy chain complementarity determining regions (CDR) are light blue (CDR H1), blue (CDR H2) and dark blue (CDR H3), while the light chain CDRs are cyan (CDR L2), green (CDR L2) and pale yellow (CDR L3). The main interacting regions are in CDR H2, CDR H3 and CDR L2. (Right) DPP4 is shown in ribbon representation (green) with Asn 229 and the attached N-glycan (yellow) shown as sticks. The RBD is oriented as shown in the left panel. (b) Antibody:RBD and DPP4:RBD crystal structure complexes. RBD in surface representation is shown with the D12 heavy and light chain binding region coloured blue and green, respectively. The CDR loops are shown as ribbons and coloured as in (a; left). The rotated RBD shows the full D12 paratope: D12 CDR H2 interacts with RBD W535 and E536 residues, which predominantly interact with the Asn 229-associated N-glycan on DPP4 (centre). RBD, shown in surface representation with the DPP4-interacting region coloured green. Major interacting regions of DPP4 are shown as ribbon representations with Asn 229 and N-glycan shown as sticks (Supplementary Fig S11; right). (c) D12 and RBD interface. All CDRs are shown in ribbon representation, with interacting residues shown as sticks, and hydrogen bonds represented by dotted lines. (d,e) Crystal structure of MERS England1 RBD and effect of critical RBD mutations on binding. RBD residues 506 and 509, identified by mutagenesis analysis, are highlighted in green. Critical RBD residues 532, 535 and 536, identified by structural definition and viral resistance evolution, reduce or eliminate D12 binding (shown in red). ELISA results show that this set of mutations eliminate F11 or D12 binding.

Mentions:
Although the two most potent neutralizing mAbs—D12 and F11—targeted the RBD, their neutralization profiles were different, when mAb neutralization was tested against the panel of eight pseudotyped reporter viruses (Fig. 1c). Notably, F11 was unable to neutralize the Bisha1 strain (GenBank ID: KF600620.1) of MERS-CoV (Supplementary Fig. 9b), which differs from other strains by an aspartic acid to glycine substitution at residue 509, rendering it resistant to F11 while still susceptible to D12 neutralization. This finding was recapitulated in a pseudotyped virus neutralization assay where F11 neutralization activity against wild-type England1 was ablated with the introduction of a D509G mutation (Supplementary Fig. 9b). D12, in contrast, neutralized both viruses irrespective of the amino-acid change at position 509. In addition, the RBD 509G mutation abrogated F11 binding using enzyme-linked immunosorbent assay (ELISA) but did not affect D12 binding (Fig. 4e).

Bottom Line:
Efforts focused solely on the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein may not optimize neutralizing antibody (NAb) responses.Here we show that immunogens based on full-length S DNA and S1 subunit protein elicit robust serum-neutralizing activity against several MERS-CoV strains in mice and non-human primates.Multiple neutralization mechanisms were demonstrated by solving the atomic structure of a NAb-RBD complex, through sequencing of neutralization escape viruses and by constructing MERS-CoV S variants for serological assays.